Head and neck squamous cell carcinoma (HNSCC) is the ninth most common cancer worldwide. In the US there are 40-50,000 new cases a year and ~12,000 deaths due to HNSCC. Early stage disease is treated with surgery or radiotherapy alone, while advanced stage HNSCC is treated with a combination of cisplatin and radiotherapy. The overall survival rate is only ~40%, and ~30% of advanced stage disease has locoregional recurrence. Tumor cells living in low oxygen microenvironments are more resistant to radiotherapy. Combining cisplatin with radiation has improved cell killing, but cisplatin is very toxic and tumors can become resistant to cisplatin during treatment. Therefore there is a need to develop new complementary therapies to improve cell killing by radiation and cisplatin. This proposal aims to determine whether disruption of mitochondrial double strand break (DSB) repair can be used as a complementary treatment to improve the therapeutic outcome of radiotherapy, or cisplatin and radiotherapy, and to develop a potential new molecular tool that can be used to enhance HNSCC cell killing. Radiotherapy and cisplatin work by damaging the cell's DNA: radiotherapy introduces DSBs, and cisplatin introduces DNA crosslinks that can be converted to DSBs during repair or by stalling replication forks. Damage to the mitochondrial genome can result in loss of functional mitochondria, an induction of oxidative stress and greater nuclear DNA damage. Both nuclear and mitochondrial DNA repair mechanisms exist, although less is known about mitochondrial repair. This work aims to understand more about cell death from mitochondrial damage. We have developed a mitochondrial-targeted bacterial Ku protein (cKumyc) that can bind DSBs but is missing the domains required to link with other human DNA repair proteins. We hypothesize that the cKumyc when targeted to the mitochondria in HNSCC cells will bind to DSBs following treatment with ionizing radiation or cisplatin, disrupting repair, causing mitochondrial genome fragmentation, reactive oxygen species (ROS) production and cell death. In stable HNSCC cell lines will be generated that can be induced with doxycycline to express cKumyc. Assays will be performed to determine clonogenic cell survival, mitochondrial function, ROS production, mitochondrial DNA fragmentation and mode of cell death after treatment with radiation and/ or cisplatin. Disruption of mitochondrial DSB repair is expected to enhance cell death. Since hypoxia plays a Specific Aim 1 significant role in resistance to radiotherapy, in we will develop a cKumyc that is expressed only in hypoxic cells and its function will be tested using the assays in aim 1 at 1-5% oxygen. This will aid future targeting of radioresistant hypoxic tumor cells. We hypothesize that cKumyc will radiosensitize the HNSCC cells under hypoxia. These proof-of-principle experiments have the potential of uncovering a new target (mitochondrial DNA) as well as a new tool for the design of a novel complementary treatment for radiotherapy/ combined chemo-radiotherapy to enhance cancer cell killing. Specific Aim 2